SUMMARY Skin protects our body against the outer environment, and its ability to repair upon injury is directly connected to both disease and survival. Failure to properly repair injured tissue can result in severe damage, such as the formation of chronic wounds, which are associated with severe complications and even death. Approximately 6.5 million people in the US suffer from chronic non-healing skin wounds, such as diabetic ulcers. At least US$25 billion is spent annually to treat chronic wounds, and this cost is skyrocketing due to increasing health care costs, an aging population and the growing rates of diabetes, cancer and obesity. Thus, chronic wounds represent a substantial burden on public health and the health care system. We still lack fundamental knowledge of distinct cellular behaviors that are coordinated to achieve tissue regeneration and repair. The goal of this proposal is to unravel how cell behaviors are properly orchestrated on the single-cell and tissue-scale level during repair. The critical barrier to addressing these fundamental questions lies in the inability to study these dynamic processes in an intact mammal. Further, interrogating skin repair is complicated by the coexistence epithelial stem cells intermixed with resident immune cells, both of which are implicated in the repair process. To this end, my laboratory has established an in vivo strategy to directly visualize and manipulate epithelial cells and immune cells in the skin epithelium of live mice, taking advantage of its unique accessibility, continuous regeneration throughout its lifetime, and efficient repair. We utilize a novel, non-invasive two-photon imaging approach to follow epithelial and immune cells during the repair process. We combine intravital microscopy with methods that we developed to manipulate distinct epithelial cell repair behaviors or resident cell types in vivo. This integrated approach allows us to dissect the coordination and functional significance of distinct cell activities, populations and interactions during repair. Thus, we have begun to understand the complex interplay between distinct epithelial cell behaviors, and between distinct cell types within the epidermis, that contribute to wound closure. The goal of this proposal is to advance our understanding of how cell populations and behaviors are orchestrated to achieve wound repair in skin, by using an integrated approach of cutting edge imaging technology, genetic manipulation and cell biology. Given that many aspects of wound repair are widely conserved in other organs, our findings will be relevant to other tissues as well, and will provide an important foundation to improve wound repair in a variety of patients.